1. Field of the Invention
The present invention relates to recombinant DNA molecules for the attachment glycoprotein gene of the avian Metapneumovirus (Colorado) (APV/CO) as well as to the corresponding protein and peptides derived therefrom. The invention further relates to the production and use of the DNA and glycoprotein in diagnostics and for vaccine production.
2. Description of the Related Art
Avian Metapneumovirus (APV) causes turkey rhinotracheitis (TRT) and is associated with swollen head syndrome (SHS) of chickens that is usually accompanied by secondary bacterial infections that increase mortality. Clinically, the disease is similar to Bordetella avium infection and is primarily respiratory in turkeys. There is loss of egg production in laying flocks. In chickens, APV infections may be subclinical, without development of SHS. The virus was first reported in South Africa during the early 1970s and virus were subsequently isolated in Europe, Israel, and Asia (Alexander, In: Diseases of Poultry, , 10th edition, Calnick B W, Barnes H J, McDougall L R, Saif Y M, and Beard C W (eds), Iowa State University Press, 541-569, 1997); Jones, Avian Pathology, 25, 639-648, 1996). During February 1997, APV was officially isolated by the National Veterinary Services Laboratory (NVSL, APHIS, USDA) from commercial turkeys in Colorado (APV/CO) following an outbreak of TRT the previous year. During the first 10 months of the U.S. outbreak it was not possible to detect virus serologically due to the lack of cross-reactivity of the United States (US) APV isolate with reagents produced in Europe. An ELISA was developed by NVSL using inactivated purified APV/CO as an antigen and serological evidence of APV infection was subsequently demonstrated in turkey flocks in the north-central United States of America (USA). In the USA mortality due to APV infections in turkeys ranges from 0 to 30% when accompanied by bacterial infections with condemnations due to airsacculitis (Senne et al., In: Proceedings, 134th Annual Conference, Schaumburg Illinois American Veterinary Medical Association, page 190, 1997). Absence of serologic reactivity of APV/CO-infected birds with APV serotypes A and B isolates and genomic sequence diversity clearly demonstrated emergence of new strains of this virus previously been considered exotic to North America.
The clinical signs in birds infected with APV are primarily respiratory and includes reales, sneezing and nasal discharge (Alexander, 1997 and Jones, 1996, supra). Viral antigen is primarily detected among experimentally infected chickens and turkey poults in the cilia of turbinate, tracheal and lung epithelial cells (Majo et al., Avian Diseases, 39, 887-896, 1995; Veterinary Microbiology, 52, 37-48, 1996; and Veterinary Microbiology, 57, 29-40, 1997). The presence of APV in the lungs proves that the virus is capable of infecting the lower respiratory tract. Extensive replication of APV in the turbinates causes severe rhinitis that allows infection by secondary bacterial agents (Jones et al., Avian Pathology, 17, 841-850, 1988; Majo et al., 1997 supra). Viral genomic RNA can be detected by ELISA or serum neutralization for several weeks after infection of adult turkeys (Jones et al, 1988, supra).
The diagnosis of APV infection has been accomplished primarily by the isolation of virus from clinical samples or the detection of virus-specific antibodies in serum. Primary isolation of APV is performed by viral replication in tracheal organ culture from specific pathogen-free chicken or turkey embryos (Wyeth and Alexander, IN: A Laboratory Manual of the Isolation and Identification of Avian Pathogens, 3rd edition, Pruchase H G, Arp L H, Domermuth C H, and Pearson J E (eds), Kennet Square, Pa.: American Association of Avian Pathologists, 121-123, 1989). However, the United States virus was isolated using chicken embryo fibroblast cells in culture (Senne et al, supra). Virus neutralization assays, immunofluorescence, and ELISA have been used for the serologic detection of antibodies to APV (Grant et al, Veterinary Record, 120, 279-280, 1987; O""Loan et al., Journal of Virological Methods, 25, 271-282, 1989).
An ELISA was used to search for antibodies to APV among chickens and turkeys in Canada (Heckert et al., Veterinary Record, 132, 172, 1993), using antigen from an APV strain from Europe. Because the current North American APV/CO isolate is not cross-reactive with European isolates, these sera should be evaluated again with a more appropriate antigen.
The presence of viral nucleic acids can be established by a reverse transcription-polymerase chain reaction (RT-PCR) method using oligonucleotide primers specific for the fusion protein gene of APV type A (Jing et al., Avian Pathology, 22, 771-783, 1993). However, an RT-PCR protocol using primers from within the APV nucleocapsid protein gene may prove more useful for detecting different viral subtypes (Bayon-Auboyer et al., Archives of Virology, 144, 10901-1109, 1999).
Vaccination with live, attenuated, cell cultured-adapted APV strains has been used to control disease caused by APV (Jones, 1996 supra; Jones et al., Veterinary Record, 119, 599-600, 1986). Currently, APV type A vaccines protect against challenge from type A and B viruses from Europe (Cook et al., Veterinary Record, 136, 392-393, 1995; Avian Pathology, 25, 231-243, 1996), although maternal antibody fails to protect pouts from challenge (Naylor et al., Avian Diseases, 41, 968-971, 1997). Use of live virus vaccine followed by inactivated vaccines provided protection against both respiratory infection and reduced egg production in turkeys (Cook et al., 1996, supra). There are no published reports demonstrating whether these vaccines will protect against disease caused by the United States isolate. Also, prototype live attenuated APV vaccines may cause disease in young poults that is apparently due to virulent viral subpopulations that must be removed from current vaccine preparations (Naylor and Jones, Vaccine 12, 1225-1230, 1994). A fowlpox virus recombinant containing the F protein gene conferred only a partial protective immune response to APV challenge (Qingzhong et al., Vaccine, 12, 569-573, 1994). Cyclophosphamide treatment of poultry to suppress B-cell responses before APV vaccination, still resulted in only a partial response to challenge. It was concluded that cellular immune responses maybe more important than humoral responses in vaccination with APV (Jones et al., Research in Veterinary Science, 53, 38-41, 1992).
Pneumovirus are members of the family Paramyxoviridae and contain a non-segmented, single-strand, negative-sense RNA genome of approximately 15 kilobases in length. Viruses related to APV include the human, bovine, ovine and caprine respiratory synitial viruses, and pneumonia virus of mice as well as the recently identified human Metapneumovirus (van den Hoogen et al., Nat. Med., Volume 7(6), 719-724, 2001). Pneumoviruses generally encode ten genes versus the six or seven of other paramyxoviruses, such as Newcastle disease virus (NDV). These include genes for the non-structural proteins (NS1 and NS2), nucleoprotein (N), phosphoprotein (P), matrix protein (M), small hydrophobic protein (SH), surface glycoprotein (G), fusion protein (F), second matrix protein (M2), and a viral RNA-dependent RNA polymerase (L). The pneumoviruses have an F protein that promotes cell fusion, these viruses do not hemagglutinate, nor do they have neuraminidase activity in their G attachment protein. This is an important distinguishing characteristic from the other paramyxoviruses (Collins et al., In: Field""s Virology, 3rd edition, Fields B N, Knipe D M, and Howley P M (eds), Philadelphia: Lippincott-Raven, 1313-1351, 1996).
Classification of European APV isolates was initially based on the molecular characterization of the virion (Collins et al., Veterinary Record, 119, 606, 1986; Collins and Gough, Journal of General Virology, 69, 909-916, 1988); electrophoretic mobility of viral proteins (Ling and Pringle, Journal of General Virology, 69, 917-923, 1988) and number of MRNA species detected in virus-infected cells (Cavanaugh and Barrett, Virus Research, 11, 241-256, 1988). Sequence information for the N (Li et al., Virus Research, 41, 185-191, 1996), P (Ling et al., Virus Research, 36, 247-257, 1995), M (Randhawa et al., Journal of General Virology, 77, 3047-3051, 1996; Yu et al., Virology, 186, 426-434, 1992), M2 (Yu et al., Journal of General Virology, 73, 1355-1356, 1992), SH (Ling et al., Journal of General Virology, 73, 1709-1715, 1992), G (Juhasz and Easton, Journal of General Virology, 75, 2873-2880, 1994; Ling et al., 1992, supra), and L (Randhawa et al., supra) protein genes is now published for several European APV isolates. The sequence in every case is most similar to other members of the Pneumovirus genus. The putative gene order of APV (3xe2x80x2-N-P-M-F-M2-SH-G-L-5xe2x80x2) is different from its mammalian counterpart (3xe2x80x2-NS1-NS2-N-P-M-SH-G-F-M2-L-5xe2x80x2), wherein the SH and G genes are located 5xe2x80x2 to the M2 gene (Ling et al., 1992, supra). The extreme 3xe2x80x2 and 5xe2x80x2 ends of one European APV isolate""s genome were determined which established that the NS1 and NS2 genes are absent in the avian viruses (Randhawa et al., Journal of Virology, 71, 9849-9854, 1997). This is different from their mammalian counterparts and along with its smaller L gene, results in APV having a genome of only 13.3 kilobases (Randhawa et al., 1996b, supra). Since APV has no NS1 or NS2 gene, but has an M2 gene; with structural characteristics like those of other pneumoviruses, it has become the type of virus of a new genus within the metapneumovirus (Pringle, Archives of Virology, 141, 2251-2256, 1996).
The G gene of APV encodes the surface glycoprotein (cell attachment protein) protein and serves as one of the major antigenic determinants of pneumoviruses. On the basis of the nucleotide sequence and the predicted amino acid sequence of the G protein gene, two APV subgroups, designated A and B, have been identified (Juhasz and Eastoh, 1994, supra). The group A viruses included isolates from the UK and France, while group B viruses included isolates from Spain, Italy, and Hungary. However, Type B are now found in the UK (Naylor et al., Avian Pathology, 26, 327-338, 1997a). The G proteins are 98.5-99.7% similar among groups, but only 38% similar between the two APV clusters. This correlates with earlier data demonstrating that various APV isolates were antigenically similar but could be separated serologically into two separate groups (Collins et al., Avian Pathology, 22, 469-479, 1993; Cook et al., Avian Pathology 22, 257-273, 1993). This relationship was confirmed by sequence analysis of the F protein gene (Naylor et al., Journal of General Virology, 79, 1393-1398, 1998) and the more conserved M gene (Randhawa et al, Virus Genes, 12, 179-183 1996a). This difference in viral proteins could account for discrepancies found when different antigens were used for serology (Eterradossi et al., Veterinary Record, 131, 563-564, 1992).
The M protein gene is highly conserved among paramyxoviruses (Rima et al., In: Genetics and Pathogenicity of Negative Strand Viruses, Kolakofsky D, and Maby B M J (eds), 254-263, 1989) and can be reliably used for molecular epidemiology (Seal et al., Virus Research, 66, 1-22, 2000a). The predicted M proteins of European APV type A and B isolates share 89% identity in their amino acid sequence. However, the predicted M protein of APV/CO shares only 78% identity with APV type A and 77% with APV type B protein sequences. The US isolate is phylogenetically separate from the European APV type A and B strains, which cluster together. Sequence information for the APV/CO M protein gene and the predicted amino acid sequence of the M protein confirm the unique nature of this isolate compared with its European counterparts (Seal, Virus Research, 58, 45-52, 1998).
More recently, the F protein gene sequence of the Colorado virus and several isolates from Minnesota have been reported (Seal et al, Virus Research, 66, 139-147, 2000b). The predicted amino acid sequence of the F protein of the US APV isolate shared approximately 71.5% sequence identity with that of the European APV types. In contrast, the European type A and B viruses shared 83% of their F protein predicted amino acid sequences with and were phylogenetically separate from the US viruses. Variation in the F protein was extensive in the first ten amino acids and the last 50 carboxy-terminal residues of the F protein (Seal et al., 2000b, supra). Cleavage of the F protein into a larger F1 and a smaller F2 protein mediates cell fusion and is necessary for the spread of the virus (Collins et al., 1996, supra). The amino-terminal portion of F1 is presumed to be involved in membrane fusion and is highly conserved among all three APV types (Naylor et al, 199, supra; Seal et al., 2000b, supra)
The cleavage site sequence of the fusion protein is highly variable among the three APV types, and codon usage for the shared amino acids is not consistent among them at these residues (Seal et al., 2000b, supra). The US isolates have a fusion protein sequence of Arg-Lys-Ala-Arg compared with Arg-Arg-Arg-Arg for the type A isolates and Arg-Lys-Lys-Arg for the type B viruses. Cleavage of the paramyxovirus F proteins required for the replication of these virus type (Nagai, Microbiology and Immunology, 39, 1-9, 1995)occurs by the action.of cellular proteases that recognize the sequence Arg-X-Arg/Lys-Arg-Arg (Hosaka et al., Journal of Biological Chemistry, 266, 12127-12130, 1991). The Ala residue present in the F cleavage site sequence of the US APV isolates but not in APV types A and B may affect their relative virulences. This could explain the lack of ciliostasis in tracheal organ cultures among US viruses (Cook et al., Avian Pathology, 28, 607-617, 1999). Also, it is possible that bacterial proteases (Akaike et al., Journal of Virology, 63, 2252-2259, 1989) play a role in F protein cleavage because severe disease is associated with secondary bacterial infections (Majo et al., 1997, supra).
The origin of the US virus and how APV emerged in North America is unknown. Viruses isolated in South America are reported to be of the APV type A (Dani et al., Avian Pathology, 28, 473-476, 1999a; Dani et al., Journal of Virological Methods, 79, 237-241, 1999b; Toro et al., Avian Diseases, 42, 815-817, 1998). Monospecific antisera and neutralizing monoclonal antibodies to APV types A and B do not neutralize the US virus and vice versa (Cook et al., 1999, supra). However, hyperimmune sera to type A virus can partially neutralize the US virus, whereas type B hyperimmune sera does not. Also, vaccination of birds with the US virus reduces signs of disease after challenge with the APV type A but not type B (Cook et al., 1999, supra). The APV isolate from the US is phylogenetically separate from the A and B types on the basis of the nucleotide sequences of the M and F amino acid coding sequences. There are fewer sequence differences between the type A virus and the US isolate than between the B type and the US isolate. It is conceivable that the US viruses could have the A type virus as a progenitor. However, the absolute distances from the A or B types to the US virus are substantial and the US virus is phylogenetically separate from the other types with very high bootstrap confidence limits. The APV isolates from the US are serologically distinct from type A and B viruses (Cook et al., 1999, supra; Senne et al., 1997, supra), and this relationship has been confirmed by sequence analysis of the genes for the M (Seal, 1998, supra) and F (Seal et al, 2000b, supra).
While various avian pneumoviruses are known to cause upper respiratory disease in avians, there remains a need for methods for detecting different strains of avian pneumoviruses in avians, especially poultry, which overcome some of the limitations of related art detection methods. The present invention described below is a novel nucleic acid encoding the Avian Metapneumovirus (Colorado) type C attachment glycoprotein DNA, a novel attachment glycoprotein and peptides derived therefrom encoded by the type C attachment glycoprotein gene, vaccines produced using said DNA, and immunodiagnostics for detecting type C avian Metapneumovirus.
It is, therefore, an object of the present invention to provide a novel nucleotide sequence encoding Avian Metapneumovirus (Colorado) Type C strain attachment glycoprotein.
A further object of the present invention is to provide an amino acid sequence for Avian Metapneumovirus Type C strain attachment glycoprotein.
Another object of the present invention is to provide methods for detecting Avian Metapneumovirus (Colorado) type C strain in animals, especially poultry using antibodies, primers, or probes produced from the novel nucleotide sequence and/or amino acid sequence for avian Metapneumovirus (Colorado) Type C strain attachment glycoprotein.
A still further object of the present invention is to provide a vaccine using the type C strain glycoprotein and/or the type C strain glycoprotein nucleic acid sequence.
Further objects and advantages of the present invention will become apparent from following description.
A plasmid containing the G attachment glycoprotein gene of avian metapneumovirus (Colorado) has been deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (P.O. Box 1549, Manassas, Va. 20108 USA) on The Accession Number is ATCC